Generation of plants with altered oil content

ABSTRACT

The present invention is directed to plants that display an altered oil content phenotype due to altered expression of a HIO32.2 nucleic acid. The invention is further directed to methods of generating plants with an altered oil content phenotype.

REFERENCE TO RELATED APPLICATIONS

This is the Continuation of application Ser. No. 10/583,199, filed onJun. 15, 2006, issued as U.S. Pat. No. 7,541,516, on Jun. 2, 2009, whichis the §371 U.S. National Stage of International Application No.PCT/US2004/042749, filed on Dec. 17, 2004, which was published inEnglish under PCT Article 21(2), and which in turn claims the benefit ofU.S. Provisional Patent Application No. 60/530,878, filed Dec. 17, 2003.All of these applications are hereby incorporated by reference.

BACKGROUND OF THE INVENTION

The ability to manipulate the composition of crop seeds, particularlythe content and composition of seed oils, has important applications inthe agricultural industries, relating both to processed food oils and tooils for animal feeding. Seeds of agricultural crops contain a varietyof valuable constituents, including oil, protein and starch. Industrialprocessing can separate some or all of these constituents for individualsale in specific applications. For instance, nearly 60% of the U.S.soybean crop is crushed by the soy processing industry. Soy processingyields purified oil, which is sold at high value, while the remainder issold principally for lower value livestock feed (U.S. Soybean Board,2001 Soy Stats). Canola seed is crushed to produce oil and theco-product canola meal (Canola Council of Canada). Nearly 20% of the1999/2000 U.S. corn crop was industrially refined, primarily forproduction of starch, ethanol and oil (Corn Refiners Association). Thus,it is often desirable to maximize oil content of seeds. For instance,for processed oilseeds such as soy and canola, increasing the absoluteoil content of the seed will increase the value of such grains. Forprocessed corn it may be desired to either increase or decrease oilcontent, depending on utilization of other major constituents.Decreasing oil may improve the quality of isolated starch by reducingundesired flavors associated with oil oxidation. Alternatively, inethanol production, where flavor is unimportant, increasing oil contentmay increase overall value. In many fed grains, such as corn and wheat,it is desirable to increase seed oil content, because oil has higherenergy content than other seed constituents such as carbohydrate.Oilseed processing, like most grain processing businesses, is acapital-intensive business; thus small shifts in the distribution ofproducts from the low valued components to the high value oil componentcan have substantial economic impacts for grain processors.

Biotechnological manipulation of oils can provide compositionalalteration and improvement of oil yield. Compositional alterationsinclude high oleic soybean and corn oil (U.S. Pat. Nos. 6,229,033 and6,248,939), and laurate-containing seeds (U.S. Pat. No. 5,639,790),among others. Work in compositional alteration has predominantly focusedon processed oilseeds but has been readily extendable to non-oilseedcrops, including corn. While there is considerable interest inincreasing oil content, the only currently practiced biotechnology inthis area is High-Oil Corn (HOC) technology (DuPont, U.S. Pat. No.5,704,160). HOC employs high oil pollinators developed by classicalselection breeding along with elite (male-sterile) hybrid females in aproduction system referred to as TopCross. The TopCross High Oil systemraises harvested grain oil content in maize from about 3.5% to about 7%,improving the energy content of the grain.

While it has been fruitful, the HOC production system has inherentlimitations. First, the system of having a low percentage of pollinatorsresponsible for an entire field's seed set contains inherent risks,particularly in drought years. Second, oil contents in current HOCfields have plateaued at about 9% oil. Finally, high-oil corn is notprimarily a biochemical change, but rather an anatomical mutant(increased embryo size) that has the indirect result of increasing oilcontent. For these reasons, an alternative high oil strategy,particularly one that derives from an altered biochemical output, wouldbe especially valuable.

The most obvious target crops for the processed oil market are soy andrapeseed, and a large body of commercial work (e.g., U.S. Pat. No.5,952,544; PCT application WO9411516) demonstrates that Arabidopsis isan excellent model for oil metabolism in these crops. Biochemicalscreens of seed oil composition have identified Arabidopsis genes formany critical biosynthetic enzymes and have led to identification ofagronomically important gene orthologs. For instance, screens usingchemically mutagenized populations have identified lipid mutants whoseseeds display altered fatty acid composition (Lemieux et al., 1990;James and Dooner, 1990). T-DNA mutagenesis screens (Feldmann et al.,1989) that detected altered fatty acid composition identified the omega3 desaturase (FAD3) and delta-12 desaturase (FAD2) genes (U.S. Pat. No.5,952,544; Yadav et al., 1993; Okuley et al., 1994). A screen whichfocused on oil content rather than oil quality, analyzedchemically-induced mutants for wrinkled seeds or altered seed density,from which altered seed oil content was inferred (Focks and Benning,1998). Another screen, designed to identify enzymes involved inproduction of very long chain fatty acids, identified a mutation in thegene encoding a diacylglycerol acyltransferase (DGAT) as beingresponsible for reduced triacyl glycerol accumulation in seeds (KatavicV et al, 1995). It was further shown that seed-specific over-expressionof the DGAT cDNA was associated with increased seed oil content (Jako etal., 2001).

Activation tagging in plants refers to a method of generating randommutations by insertion of a heterologous nucleic acid constructcomprising regulatory sequences (e.g., an enhancer) into a plant genome.The regulatory sequences can act to enhance transcription of one or morenative plant genes; accordingly, activation tagging is a fruitful methodfor generating gain-of-function, generally dominant mutants (see, e.g.,Hayashi et al., 1992; Weigel D et al. 2000). The inserted constructprovides a molecular tag for rapid identification of the native plantwhose mis-expression causes the mutant phenotype. Activation tagging mayalso cause loss-of-function phenotypes. The insertion may result indisruption of a native plant gene, in which case the phenotype isgenerally recessive.

Activation tagging has been used in various species, including tobaccoand Arabidopsis, to identify many different kinds of mutant phenotypesand the genes associated with these phenotypes (Wilson et al., 1996,Schaffer et al., 1998, Fridborg et al., 1999; Kardailsky et al., 1999;Christensen S et al. 1998).

SUMMARY OF THE INVENTION

The invention provides a transgenic plant having a high oil phenotype.The transgenic plant comprises a transformation vector comprising anucleotide sequence that encodes or is complementary to a sequence thatencodes a HIO32.2 polypeptide comprising the amino acid sequence of SEQID NO:2, or an ortholog thereof. In preferred embodiments, thetransgenic plant is selected from the group consisting of rapeseed, soy,corn, sunflower, cotton, cocoa, safflower, oil palm, coconut palm, flax,castor and peanut. The invention also provides a method of producing oilcomprising growing the transgenic plant and recovering oil from saidplant. The invention further provides a method of generating a planthaving a high oil phenotype by identifying a plant that has an allele inits HIO32.2 gene that results in increased oil content compared toplants lacking the allele and generating progeny of the identifiedplant, wherein the generated progeny inherit the allele and have thehigh oil phenotype.

The transgenic plant of the invention is produced by a method thatcomprises introducing into progenitor cells of the plant a planttransformation vector comprising a nucleotide sequence that encodes oris complementary to a sequence that encodes a HIO32.2 polypeptide, andgrowing the transformed progenitor cells to produce a transgenic plant,wherein the HIO32.2 polynucleotide sequence is expressed causing thehigh oil phenotype.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

Unless otherwise indicated, all technical and scientific terms usedherein have the same meaning as they would to one skilled in the art ofthe present invention. Practitioners are particularly directed toSambrook et al., 1989, and Ausubel F M et al., 1993, for definitions andterms of the art. It is to be understood that this invention is notlimited to the particular methodology, protocols, and reagentsdescribed, as these may vary.

As used herein, the term “vector” refers to a nucleic acid constructdesigned for transfer between different host cells. An “expressionvector” refers to a vector that has the ability to incorporate andexpress heterologous DNA fragments in a foreign cell. Many prokaryoticand eukaryotic expression vectors are commercially available. Selectionof appropriate expression vectors is within the knowledge of thosehaving skill in the art.

A “heterologous” nucleic acid construct or sequence has a portion of thesequence that is not native to the plant cell in which it is expressed.Heterologous, with respect to a control sequence refers to a controlsequence (i.e. promoter or enhancer) that does not function in nature toregulate the same gene the expression of which it is currentlyregulating. Generally, heterologous nucleic acid sequences are notendogenous to the cell or part of the genome in which they are present,and have been added to the cell, by infection, transfection,microinjection, electroporation, or the like. A “heterologous” nucleicacid construct may contain a control sequence/DNA coding sequencecombination that is the same as, or different from a controlsequence/DNA coding sequence combination found in the native plant.

As used herein, the term “gene” means the segment of DNA involved inproducing a polypeptide chain, which may or may not include regionspreceding and following the coding region, e.g. 5′ untranslated (5′ UTR)or “leader” sequences and 3′ UTR or “trailer” sequences, as well asintervening sequences (introns) between individual coding segments(exons) and non-transcribed regulatory sequence.

As used herein, “recombinant” includes reference to a cell or vector,that has been modified by the introduction of a heterologous nucleicacid sequence or that the cell is derived from a cell so modified. Thus,for example, recombinant cells express genes that are not found inidentical form within the native (non-recombinant) form of the cell orexpress native genes that are otherwise abnormally expressed, underexpressed or not expressed at all as a result of deliberate humanintervention.

As used herein, the term “gene expression” refers to the process bywhich a polypeptide is produced based on the nucleic acid sequence of agene. The process includes both transcription and translation;accordingly, “expression” may refer to either a polynucleotide orpolypeptide sequence, or both. Sometimes, expression of a polynucleotidesequence will not lead to protein translation. “Over-expression” refersto increased expression of a polynucleotide and/or polypeptide sequencerelative to its expression in a wild-type (or other reference [e.g.,non-transgenic]) plant and may relate to a naturally-occurring ornon-naturally occurring sequence. “Ectopic expression” refers toexpression at a time, place, and/or increased level that does notnaturally occur in the non-altered or wild-type plant.“Under-expression” refers to decreased expression of a polynucleotideand/or polypeptide sequence, generally of an endogenous gene, relativeto its expression in a wild-type plant. The terms “mis-expression” and“altered expression” encompass over-expression, under-expression, andectopic expression.

The term “introduced” in the context of inserting a nucleic acidsequence into a cell, means “transfection”, or “transformation” or“transduction” and includes reference to the incorporation of a nucleicacid sequence into a eukaryotic or prokaryotic cell where the nucleicacid sequence may be incorporated into the genome of the cell (forexample, chromosome, plasmid, plastid, or mitochondrial DNA), convertedinto an autonomous replicon, or transiently expressed (for example,transfected mRNA).

As used herein, a “plant cell” refers to any cell derived from a plant,including cells from undifferentiated tissue (e.g., callus) as well asplant seeds, pollen, progagules and embryos.

As used herein, the terms “native” and “wild-type” relative to a givenplant trait or phenotype refers to the form in which that trait orphenotype is found in the same variety of plant in nature.

As used herein, the term “modified” regarding a plant trait, refers to achange in the phenotype of a transgenic plant relative to the similarnon-transgenic plant. An “interesting phenotype (trait)” with referenceto a transgenic plant refers to an observable or measurable phenotypedemonstrated by a T1 and/or subsequent generation plant, which is notdisplayed by the corresponding non-transgenic (i.e., a genotypicallysimilar plant that has been raised or assayed under similar conditions).An interesting phenotype may represent an improvement in the plant ormay provide a means to produce improvements in other plants. An“improvement” is a feature that may enhance the utility of a plantspecies or variety by providing the plant with a unique and/or novelquality. An “altered oil content phenotype” refers to measurablephenotype of a genetically modified plant, where the plant displays astatistically significant increase or decrease in overall oil content(i.e., the percentage of seed mass that is oil), as compared to thesimilar, but non-modified plant. A high oil phenotype refers to anincrease in overall oil content.

As used herein, a “mutant” polynucleotide sequence or gene differs fromthe corresponding wild type polynucleotide sequence or gene either interms of sequence or expression, where the difference contributes to amodified plant phenotype or trait. Relative to a plant or plant line,the term “mutant” refers to a plant or plant line which has a modifiedplant phenotype or trait, where the modified phenotype or trait isassociated with the modified expression of a wild type polynucleotidesequence or gene.

As used herein, the term “T1” refers to the generation of plants fromthe seed of T0 plants. The T1 generation is the first set of transformedplants that can be selected by application of a selection agent, e.g.,an antibiotic or herbicide, for which the transgenic plant contains thecorresponding resistance gene. The term “T2” refers to the generation ofplants by self-fertilization of the flowers of T1 plants, previouslyselected as being transgenic. T3 plants are generated from T2 plants,etc. As used herein, the “direct progeny” of a given plant derives fromthe seed (or, sometimes, other tissue) of that plant and is in theimmediately subsequent generation; for instance, for a given lineage, aT2 plant is the direct progeny of a T1 plant. The “indirect progeny” ofa given plant derives from the seed (or other tissue) of the directprogeny of that plant, or from the seed (or other tissue) of subsequentgenerations in that lineage; for instance, a T3 plant is the indirectprogeny of a T1 plant.

As used herein, the term “plant part” includes any plant organ ortissue, including, without limitation, seeds, embryos, meristematicregions, callus tissue, leaves, roots, shoots, gametophytes,sporophytes, pollen, and microspores. Plant cells can be obtained fromany plant organ or tissue and cultures prepared therefrom. The class ofplants which can be used in the methods of the present invention isgenerally as broad as the class of higher plants amenable totransformation techniques, including both monocotyledenous anddicotyledenous plants.

As used herein, “transgenic plant” includes a plant that compriseswithin its genome a heterologous polynucleotide. The heterologouspolynucleotide can be either stably integrated into the genome, or canbe extra-chromosomal. Preferably, the polynucleotide of the presentinvention is stably integrated into the genome such that thepolynucleotide is passed on to successive generations. A plant cell,tissue, organ, or plant into which the heterologous polynucleotides havebeen introduced is considered “transformed”, “transfected”, or“transgenic”. Direct and indirect progeny of transformed plants or plantcells that also contain the heterologous polynucleotide are alsoconsidered transgenic.

Identification of Plants with an Altered Oil Content Phenotype

We used an Arabidopsis activation tagging screen to identify theassociation between the gene we have designated “HIO32.2,” (At3g47710;GI#22331644 encoding protein T23J7.40 (GI#22331654), and an altered oilcontent phenotype (specifically, a high oil phenotype). Briefly, and asfurther described in the Examples, a large number of Arabidopsis plantswere mutated by transformation with the pSKI015 vector, which comprisesa T-DNA from the Ti plasmid of Agrobacterium tumifaciens, a viralenhancer element, and a selectable marker gene (Weigel et al, 2000).When the T-DNA inserts into the genome of transformed plants, theenhancer element can cause up-regulation genes in the vicinity,generally within about 10 kilobase (kb) of the enhancers. To identifytransgenic plants, T1 plants were exposed to the selective agent inorder to specifically recover transformed plants that expressed theselectable marker and therefore harboring the T-DNA. T2 seed washarvested from these plants. Lipids were extracted from of about 15-20T2 seeds. Gas chromatography (GC) analysis was performed to determinefatty acid content and composition of seed samples.

An Arabidopsis line that showed a high-oil phenotype was identified,wherein oil content (i.e., fatty acids) constituted about 36.3% of seedmass compared to an average oil content of about 27.0% for seed fromother plants grown and analyzed at the same time (a 34% in oil). Theassociation of the HIO32.2 gene with the high oil phenotype wasdiscovered by identifying the site of T-DNA insertion, and as shown inthe Examples, demonstrating genetic co-segregation of the high seed oilphenotype and the presence of the T-DNA. Accordingly, HIO32.2 genesand/or polypeptides may be employed in the development of geneticallymodified plants having a modified oil content phenotype (“a HIO32.2phenotype”). HIO32.2 genes may be used in the generation of oilseedcrops that provide improved oil yield from oilseed processing and in thegeneration of feed grain crops that provide increased energy for animalfeeding. HIO32.2 genes may further be used to increase the oil contentof specialty oil crops, in order to augment yield of desired unusualfatty acids. Transgenic plants that have been genetically modified toexpress HIO32.2 can be used in the production of oil, wherein thetransgenic plants are grown, and oil is obtained from plant parts (e.g.seed) using standard methods.

HIO32.2 Nucleic Acids and Polypeptides

Arabidopsis HIO32.2 nucleic acid (genomic DNA) sequence is provided inSEQ ID NO:1 and in Genbank entry GI#22331644. The corresponding proteinsequence is provided in SEQ ID NO:2 and in GI#22331645. Nucleic acidsand/or proteins that are orthologs or paralogs of Arabidopsis HIO32.2,are described in Example 3 below.

As used herein, the term “HIO32.2 polypeptide” refers to a full-lengthHIO32.2 protein or a fragment, derivative (variant), or ortholog thereofthat is “functionally active,” meaning that the protein fragment,derivative, or ortholog exhibits one or more or the functionalactivities associated with the polypeptide of SEQ ID NO:2. In onepreferred embodiment, a functionally active HIO32.2 polypeptide causesan altered oil content phenotype when mis-expressed in a plant. In afurther preferred embodiment, mis-expression of the HIO32.2 polypeptidecauses a high oil phenotype in a plant. In another embodiment, afunctionally active HIO32.2 polypeptide is capable of rescuing defective(including deficient) endogenous HIO32.2 activity when expressed in aplant or in plant cells; the rescuing polypeptide may be from the sameor from a different species as that with defective activity. In anotherembodiment, a functionally active fragment of a full length HIO32.2polypeptide (i.e., a native polypeptide having the sequence of SEQ IDNO:2 or a naturally occurring ortholog thereof) retains one of more ofthe biological properties associated with the full-length HIO32.2polypeptide, such as signaling activity, binding activity, catalyticactivity, or cellular or extra-cellular localizing activity. A HIO32.2fragment preferably comprises a HIO32.2 domain, such as a C- orN-terminal or catalytic domain, among others, and preferably comprisesat least 10, preferably at least 20, more preferably at least 25, andmost preferably at least 50 contiguous amino acids of a HIO32.2 protein.Functional domains can be identified using the PFAM program (Bateman Aet al., 1999 Nucleic Acids Res 27:260-262). A preferred HIO32.2 fragmentcomprises a helix-loop-helix DNA-binding domain (PF00010). Functionallyactive variants of full-length HIO32.2 polypeptides or fragments thereofinclude polypeptides with amino acid insertions, deletions, orsubstitutions that retain one of more of the biological propertiesassociated with the full-length HIO32.2 polypeptide. In some cases,variants are generated that change the post-translational processing ofa HIO32.2 polypeptide. For instance, variants may have altered proteintransport or protein localization characteristics or altered proteinhalf-life compared to the native polypeptide.

As used herein, the term “HIO32.2 nucleic acid” encompasses nucleicacids with the sequence provided in or complementary to the sequenceprovided in SEQ ID NO:1, as well as functionally active fragments,derivatives, or orthologs thereof. A HIO32.2 nucleic acid of thisinvention may be DNA, derived from genomic DNA or cDNA, or RNA.

In one embodiment, a functionally active HIO32.2 nucleic acid encodes oris complementary to a nucleic acid that encodes a functionally activeHIO32.2 polypeptide. Included within this definition is genomic DNA thatserves as a template for a primary RNA transcript (i.e., an mRNAprecursor) that requires processing, such as splicing, before encodingthe functionally active HIO32.2 polypeptide. A HIO32.2 nucleic acid caninclude other non-coding sequences, which may or may not be transcribed;such sequences include 5′ and 3′ UTRs, polyadenylation signals andregulatory sequences that control gene expression, among others, as areknown in the art. Some polypeptides require processing events, such asproteolytic cleavage, covalent modification, etc., in order to becomefully active. Accordingly, functionally active nucleic acids may encodethe mature or the pre-processed HIO32.2 polypeptide, or an intermediateform. A HIO32.2 polynucleotide can also include heterologous codingsequences, for example, sequences that encode a marker included tofacilitate the purification of the fused polypeptide, or atransformation marker.

In another embodiment, a functionally active HIO32.2 nucleic acid iscapable of being used in the generation of loss-of-function HIO32.2phenotypes, for instance, via antisense suppression, co-suppression,etc.

In one preferred embodiment, a HIO32.2 nucleic acid used in the methodsof this invention comprises a nucleic acid sequence that encodes or iscomplementary to a sequence that encodes a HIO32.2 polypeptide having atleast 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or more sequence identityto the polypeptide sequence presented in SEQ ID NO:2.

In another embodiment a HIO32.2 polypeptide of the invention comprises apolypeptide sequence with at least 50% or 60% identity to the HIO32.2polypeptide sequence of SEQ ID NO:2, and may have at least 70%, 80%,85%, 90% or 95% or more sequence identity to the HIO32.2 polypeptidesequence of SEQ ID NO:2. In another embodiment, a HIO32.2 polypeptidecomprises a polypeptide sequence with at least 50%, 60%, 70%, 80%, 85%,90% or 95% or more sequence identity to a functionally active fragmentof the polypeptide presented in SEQ ID NO:2, such as a helix-loop-helixDNA-binding domain. In yet another embodiment, a HIO32.2 polypeptidecomprises a polypeptide sequence with at least 50%, 60%, 70%, 80%, or90% identity to the polypeptide sequence of SEQ ID NO:2 over its entirelength and comprises a helix-loop-helix DNA-binding domain.

In another aspect, a HIO32.2 polynucleotide sequence is at least 50% to60% identical over its entire length to the HIO32.2 nucleic acidsequence presented as SEQ ID NO:1, or nucleic acid sequences that arecomplementary to such a HIO32.2 sequence, and may comprise at least 70%,80%, 85%, 90% or 95% or more sequence identity to the HIO32.2 sequencepresented as SEQ ID NO:1 or a functionally active fragment thereof, orcomplementary sequences.

As used herein, “percent (%) sequence identity” with respect to aspecified subject sequence, or a specified portion thereof, is definedas the percentage of nucleotides or amino acids in the candidatederivative sequence identical with the nucleotides or amino acids in thesubject sequence (or specified portion thereof), after aligning thesequences and introducing gaps, if necessary to achieve the maximumpercent sequence identity, as generated by the program WU-BLAST-2.0a19(Altschul et al., J. Mol. Biol. (1990) 215:403-410) with searchparameters set to default values. The HSP S and HSP S2 parameters aredynamic values and are established by the program itself depending uponthe composition of the particular sequence and composition of theparticular database against which the sequence of interest is beingsearched. A “% identity value” is determined by the number of matchingidentical nucleotides or amino acids divided by the sequence length forwhich the percent identity is being reported. “Percent (%) amino acidsequence similarity” is determined by doing the same calculation as fordetermining % amino acid sequence identity, but including conservativeamino acid substitutions in addition to identical amino acids in thecomputation. A conservative amino acid substitution is one in which anamino acid is substituted for another amino acid having similarproperties such that the folding or activity of the protein is notsignificantly affected. Aromatic amino acids that can be substituted foreach other are phenylalanine, tryptophan, and tyrosine; interchangeablehydrophobic amino acids are leucine, isoleucine, methionine, and valine;interchangeable polar amino acids are glutamine and asparagine;interchangeable basic amino acids are arginine, lysine and histidine;interchangeable acidic amino acids are aspartic acid and glutamic acid;and interchangeable small amino acids are alanine, serine, threonine,cysteine and glycine.

Derivative nucleic acid molecules of the subject nucleic acid moleculesinclude sequences that selectively hybridize to the nucleic acidsequence of SEQ ID NO:1. The stringency of hybridization can becontrolled by temperature, ionic strength, pH, and the presence ofdenaturing agents such as formamide during hybridization and washing.Conditions routinely used are well known (see, e.g., Current Protocol inMolecular Biology, Vol. 1, Chap. 2.10, John Wiley & Sons, Publishers(1994); Sambrook et al., Molecular Cloning, Cold Spring Harbor (1989)).In some embodiments, a nucleic acid molecule of the invention is capableof hybridizing to a nucleic acid molecule containing the nucleotidesequence of SEQ ID NO:1 under stringent hybridization conditions thatare: prehybridization of filters containing nucleic acid for 8 hours toovernight at 65° C. in a solution comprising 6× single strength citrate(SSC) (1×SSC is 0.15 M NaCl, 0.015 M Na citrate; pH 7.0), 5×Denhardt'ssolution, 0.05% sodium pyrophosphate and 100 μg/ml herring sperm DNA;hybridization for 18-20 hours at 65° C. in a solution containing 6×SSC,1×Denhardt's solution, 100 μg/ml yeast tRNA and 0.05% sodiumpyrophosphate; and washing of filters at 65° C. for 1 h in a solutioncontaining 0.1×SSC and 0.1% SDS (sodium dodecyl sulfate). In otherembodiments, moderately stringent hybridization conditions are used thatare: pretreatment of filters containing nucleic acid for 6 h at 40° C.in a solution containing 35% formamide, 5×SSC, 50 mM Tris-HCl (pH 7.5),5 mM EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA, and 500 μg/ml denatured salmonsperm DNA; hybridization for 18-20 h at 40° C. in a solution containing35% formamide, 5×SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.02% PVP,0.02% Ficoll, 0.2% BSA, 100 μg/ml salmon sperm DNA, and 10% (wt/vol)dextran sulfate; followed by washing twice for 1 hour at 55° C. in asolution containing 2×SSC and 0.1% SDS. Alternatively, low stringencyconditions can be used that comprise: incubation for 8 hours toovernight at 37° C. in a solution comprising 20% formamide, 5×SSC, 50 mMsodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextran sulfate,and 20 μg/ml denatured sheared salmon sperm DNA; hybridization in thesame buffer for 18 to 20 hours; and washing of filters in 1×SSC at about37° C. for 1 hour.

As a result of the degeneracy of the genetic code, a number ofpolynucleotide sequences encoding a HIO32.2 polypeptide can be produced.For example, codons may be selected to increase the rate at whichexpression of the polypeptide occurs in a particular host species, inaccordance with the optimum codon usage dictated by the particular hostorganism (see, e.g., Nakamura et al, 1999). Such sequence variants maybe used in the methods of this invention.

The methods of the invention may use orthologs of the ArabidopsisHIO32.2. Methods of identifying the orthologs in other plant species areknown in the art. Normally, orthologs in different species retain thesame function, due to presence of one or more protein motifs and/or3-dimensional structures. In evolution, when a gene duplication eventfollows speciation, a single gene in one species, such as Arabidopsis,may correspond to multiple genes (paralogs) in another. As used herein,the term “orthologs” encompasses paralogs. When sequence data isavailable for a particular plant species, orthologs are generallyidentified by sequence homology analysis, such as BLAST analysis,usually using protein bait sequences. Sequences are assigned as apotential ortholog if the best hit sequence from the forward BLASTresult retrieves the original query sequence in the reverse BLAST(Huynen M A and Bork P, Proc Natl Acad Sci (1998) 95:5849-5856; Huynen MA et al., Genome Research (2000) 10:1204-1210). Programs for multiplesequence alignment, such as CLUSTAL (Thompson J D et al, 1994, NucleicAcids Res 22:4673-4680) may be used to highlight conserved regionsand/or residues of orthologous proteins and to generate phylogenetictrees. In a phylogenetic tree representing multiple homologous sequencesfrom diverse species (e.g., retrieved through BLAST analysis),orthologous sequences from two species generally appear closest on thetree with respect to all other sequences from these two species.Structural threading or other analysis of protein folding (e.g., usingsoftware by ProCeryon, Biosciences, Salzburg, Austria) may also identifypotential orthologs. Nucleic acid hybridization methods may also be usedto find orthologous genes and are preferred when sequence data are notavailable. Degenerate PCR and screening of cDNA or genomic DNA librariesare common methods for finding related gene sequences and are well knownin the art (see, e.g., Sambrook, 1989; Dieffenbach and Dveksler, 1995).For instance, methods for generating a cDNA library from the plantspecies of interest and probing the library with partially homologousgene probes are described in Sambrook et al. A highly conserved portionof the Arabidopsis HIO32.2 coding sequence may be used as a probe.HIO32.2 ortholog nucleic acids may hybridize to the nucleic acid of SEQID NO:1 under high, moderate, or low stringency conditions. Afteramplification or isolation of a segment of a putative ortholog, thatsegment may be cloned and sequenced by standard techniques and utilizedas a probe to isolate a complete cDNA or genomic clone. Alternatively,it is possible to initiate an EST project to generate a database ofsequence information for the plant species of interest. In anotherapproach, antibodies that specifically bind known HIO32.2 polypeptidesare used for ortholog isolation (see, e.g., Harlow and Lane, 1988,1999). Western blot analysis can determine that a HIO32.2 ortholog(i.e., an orthologous protein) is present in a crude extract of aparticular plant species. When reactivity is observed, the sequenceencoding the candidate ortholog may be isolated by screening expressionlibraries representing the particular plant species. Expressionlibraries can be constructed in a variety of commercially availablevectors, including lambda gt11, as described in Sambrook, et al., 1989.Once the candidate ortholog(s) are identified by any of these means,candidate orthologous sequence are used as bait (the “query”) for thereverse BLAST against sequences from Arabidopsis or other species inwhich HIO32.2 nucleic acid and/or polypeptide sequences have beenidentified.

HIO32.2 nucleic acids and polypeptides may be obtained using anyavailable method. For instance, techniques for isolating cDNA or genomicDNA sequences of interest by screening DNA libraries or by usingpolymerase chain reaction (PCR), as previously described, are well knownin the art. Alternatively, nucleic acid sequence may be synthesized. Anyknown method, such as site directed mutagenesis (Kunkel T A et al.,1991), may be used to introduce desired changes into a cloned nucleicacid.

In general, the methods of the invention involve incorporating thedesired form of the HIO32.2 nucleic acid into a plant expression vectorfor transformation of in plant cells, and the HIO32.2 polypeptide isexpressed in the host plant.

An isolated HIO32.2 nucleic acid molecule is other than in the form orsetting in which it is found in nature and is identified and separatedfrom least one contaminant nucleic acid molecule with which it isordinarily associated in the natural source of the HIO32.2 nucleic acid.However, an isolated HIO32.2 nucleic acid molecule includes HIO32.2nucleic acid molecules contained in cells that ordinarily expressHIO32.2 where, for example, the nucleic acid molecule is in achromosomal location different from that of natural cells.

Generation of Genetically Modified Plants with an Altered Oil ContentPhenotype

HIO32.2 nucleic acids and polypeptides may be used in the generation ofgenetically modified plants having a modified oil content phenotype. Asused herein, a “modified oil content phenotype” may refer to modifiedoil content in any part of the plant; the modified oil content is oftenobserved in seeds. In a preferred embodiment, altered expression of theHIO32.2 gene in a plant is used to generate plants with a high oilphenotype.

The methods described herein are generally applicable to all plants.Although activation tagging and gene identification is carried out inArabidopsis, the HIO32.2 gene (or an ortholog, variant or fragmentthereof) may be expressed in any type of plant. In a preferredembodiment, the invention is directed to oil-producing plants, whichproduce and store triacylglycerol in specific organs, primarily inseeds. Such species include soybean (Glycine max), rapeseed and canola(including Brassica napus, B. campestris), sunflower (Helianthus annus),cotton (Gossypium hirsutum), corn (Zea mays), cocoa (Theobroma cacao),safflower (Carthamus tinctorius), oil palm (Elaeis guineensis), coconutpalm (Cocos nucifera), flax (Linum usitatissimum), castor (Ricinuscommunis) and peanut (Arachis hypogaea). The invention may also bedirected to fruit- and vegetable-bearing plants, grain-producing plants,nut-producing plants, rapid cycling Brassica species, alfalfa (Medicagosativa), tobacco (Nicotiana), turfgrass (Poaceae family), other foragecrops, and wild species that may be a source of unique fatty acids.

The skilled artisan will recognize that a wide variety of transformationtechniques exist in the art, and new techniques are continually becomingavailable. Any technique that is suitable for the target host plant canbe employed within the scope of the present invention. For example, theconstructs can be introduced in a variety of forms including, but notlimited to as a strand of DNA, in a plasmid, or in an artificialchromosome. The introduction of the constructs into the target plantcells can be accomplished by a variety of techniques, including, but notlimited to Agrobacterium-mediated transformation, electroporation,microinjection, microprojectile bombardment calcium-phosphate-DNAco-precipitation or liposome-mediated transformation of a heterologousnucleic acid. The transformation of the plant is preferably permanent,i.e. by integration of the introduced expression constructs into thehost plant genome, so that the introduced constructs are passed ontosuccessive plant generations. Depending upon the intended use, aheterologous nucleic acid construct comprising an HIO32.2 polynucleotidemay encode the entire protein or a biologically active portion thereof.

In one embodiment, binary Ti-based vector systems may be used totransfer polynucleotides. Standard Agrobacterium binary vectors areknown to those of skill in the art, and many are commercially available(e.g., pBI121 Clontech Laboratories, Palo Alto, Calif.).

The optimal procedure for transformation of plants with Agrobacteriumvectors will vary with the type of plant being transformed. Exemplarymethods for Agrobacterium-mediated transformation include transformationof explants of hypocotyl, shoot tip, stem or leaf tissue, derived fromsterile seedlings and/or plantlets. Such transformed plants may bereproduced sexually, or by cell or tissue culture. Agrobacteriumtransformation has been previously described for a large number ofdifferent types of plants and methods for such transformation may befound in the scientific literature. Of particular relevance are methodsto transform commercially important crops, such as rapeseed (De Block etal., 1989), sunflower (Everett et al., 1987), and soybean (Christou etal., 1989; Kline et al., 1987).

Expression (including transcription and translation) of HIO32.2 may beregulated with respect to the level of expression, the tissue type(s)where expression takes place and/or developmental stage of expression. Anumber of heterologous regulatory sequences (e.g., promoters andenhancers) are available for controlling the expression of a HIO32.2nucleic acid. These include constitutive, inducible and regulatablepromoters, as well as promoters and enhancers that control expression ina tissue- or temporal-specific manner. Exemplary constitutive promotersinclude the raspberry E4 promoter (U.S. Pat. Nos. 5,783,393 and5,783,394), the 35S CaMV (Jones J D et al, 1992), the CsVMV promoter(Verdaguer B et al., 1998) and the melon actin promoter (published PCTapplication WO0056863). Exemplary tissue-specific promoters include thetomato E4 and E8 promoters (U.S. Pat. No. 5,859,330) and the tomato 2AIIgene promoter (Van Haaren M J J et al., 1993).

In one preferred embodiment, HIO32.2 expression is under control ofregulatory sequences from genes whose expression is associated withearly seed and/or embryo development. Legume genes whose promoters areassociated with early seed and embryo development include V. fabalegumin (Baumlein et al., 1991, Mol Gen Genet. 225:121-8; Baumlein etal., 1992, Plant J 2:233-9), V. faba usp (Fiedler et al., 1993, PlantMol Biol 22:669-79), pea convicilin (Bown et al., 1988, Biochem J251:717-26), pea lectin (dePater et al., 1993, Plant Cell 5:877-86), P.vulgaris beta phaseolin (Bustos et al., 1991, EMBO J. 10: 1469-79), P.vulgaris DLEC2 and PHS [beta] (Bobb et al, 1997, Nucleic Acids Res25:641-7), and soybean beta-Conglycinin, 7S storage protein (Chamberlandet al., 1992, Plant Mol Biol 19:937-49). Cereal genes whose promotersare associated with early seed and embryo development include riceglutelin (“GluA-3,” Yoshihara and Takaiwa, 1996, Plant Cell Physiol37:107-11; “GluB-1,” Takaiwa et al., 1996, Plant Mol Biol 30:1207-21;Washida et al., 1999, Plant Mol Biol 40:1-12; “Gt3,” Leisy et al., 1990,Plant Mol Biol 14:41-50), rice prolamin (Zhou & Fan, 1993, TransgenicRes 2:141-6), wheat prolamin (Hammond-Kosack et al., 1993, EMBO J.12:545-54), maize zein (Z4, Matzke et al., 1990, Plant Mol Biol14:323-32), and barley B-hordeins (Entwistle et al., 1991, Plant MolBiol 17:1217-31). Other genes whose promoters are associated with earlyseed and embryo development include oil palm GLO7A (7S globulin,Morcillo et al., 2001, Physiol Plant 112:233-243), Brassica napus napin,2S storage protein, and napA gene (Josefsson et al., 1987, J Biol Chem262:12196-201; Stalberg et al., 1993, Plant Mol Biol 1993 23:671-83;Ellerstrom et al., 1996, Plant Mol Biol 32:1019-27), Brassica napusoleosin (Keddie et al., 1994, Plant Mol Biol 24:327-40), Arabidopsisoleosin (Plant et al., 1994, Plant Mol Biol 25:193-205), ArabidopsisFAE1 (Rossak et al., 2001, Plant Mol Biol 46:717-25), Canavalia gladiataconA (Yamamoto et al., 1995, Plant Mol Biol 27:729-41), and Catharanthusroseus strictosidine synthase (Str, Ouwerkerk and Memelink, 1999, MolGen Genet. 261:635-43). In another preferred embodiment, regulatorysequences from genes expressed during oil biosynthesis are used (see,e.g., U.S. Pat. No. 5,952,544). Alternative promoters are from plantstorage protein genes (Bevan et al, 1993, Philos Trans R Soc Lond B BiolSci 342:209-15).

In yet another aspect, in some cases it may be desirable to inhibit theexpression of endogenous HIO32.2 in a host cell. Exemplary methods forpracticing this aspect of the invention include, but are not limited toantisense suppression (Smith, et al., 1988; van der Krol et al., 1988);co-suppression (Napoli, et al., 1990); ribozymes (PCT Publication WO97/10328); and combinations of sense and antisense (Waterhouse, et al.,1998). Methods for the suppression of endogenous sequences in a hostcell typically employ the transcription or transcription and translationof at least a portion of the sequence to be suppressed. Such sequencesmay be homologous to coding as well as non-coding regions of theendogenous sequence. Antisense inhibition may use the entire cDNAsequence (Sheehy et al., 1988), a partial cDNA sequence includingfragments of 5′ coding sequence, (Cannon et al., 1990), or 3′ non-codingsequences (Ch'ng et al., 1989). Cosuppression techniques may use theentire cDNA sequence (Napoli et al., 1990; van der Krol et al., 1990),or a partial cDNA sequence (Smith et al., (1990).

Standard molecular and genetic tests may be performed to further analyzethe association between a gene and an observed phenotype. Exemplarytechniques are described below.

1. DNA/RNA Analysis

The stage- and tissue-specific gene expression patterns in mutant versuswild-type lines may be determined, for instance, by in situhybridization. Analysis of the methylation status of the gene,especially flanking regulatory regions, may be performed. Other suitabletechniques include overexpression, ectopic expression, expression inother plant species and gene knock-out (reverse genetics, targetedknock-out, viral induced gene silencing [VIGS, see Baulcombe D, 1999]).

In a preferred application expression profiling, generally by microarrayanalysis, is used to simultaneously measure differences or inducedchanges in the expression of many different genes. Techniques formicroarray analysis are well known in the art (Schena M et al., Science(1995) 270:467-470; Baldwin D et al., 1999; Dangond F, Physiol Genomics(2000) 2:53-58; van Hal N L et al., J Biotechnol (2000) 78:271-280;Richmond T and Somerville S, Curr Opin Plant Biol (2000) 3:108-116).Expression profiling of individual tagged lines may be performed. Suchanalysis can identify other genes that are coordinately regulated as aconsequence of the overexpression of the gene of interest, which mayhelp to place an unknown gene in a particular pathway.

2. Gene Product Analysis

Analysis of gene products may include recombinant protein expression,antisera production, immunolocalization, biochemical assays forcatalytic or other activity, analysis of phosphorylation status, andanalysis of interaction with other proteins via yeast two-hybrid assays.

3. Pathway Analysis

Pathway analysis may include placing a gene or gene product within aparticular biochemical, metabolic or signaling pathway based on itsmis-expression phenotype or by sequence homology with related genes.Alternatively, analysis may comprise genetic crosses with wild-typelines and other mutant lines (creating double mutants) to order the genein a pathway, or determining the effect of a mutation on expression ofdownstream “reporter” genes in a pathway.

Generation of Mutated Plants with an Altered Oil Content Phenotype

The invention further provides a method of identifying non-transgenicplants that have mutations in or an allele of endogenous HIO32.3 thatconfer a HIO32.3 phenotype to these plants and their progeny. In onemethod, called “TILLING” (for targeting induced local lesions ingenomes), mutations are induced in the seed of a plant of interest, forexample, using EMS treatment. The resulting plants are grown andself-fertilized, and the progeny are used to prepare DNA samples.HIO32.3-specific PCR is used to identify whether a mutated plant has aHIO32.3 mutation. Plants having HIO32.3 mutations may then be tested foraltered oil content, or alternatively, plants may be tested for alteredoil content, and then HIO32.3-specific PCR is used to determine whethera plant having altered oil content has a mutated HIO32.3 gene. TILLINGcan identify mutations that may alter the expression of specific genesor the activity of proteins encoded by these genes (see Colbert et al(2001) Plant Physiol 126:480-484; McCallum et al (2000) NatureBiotechnology 18:455-457).

In another method, a candidate gene Quantitative Trait Locus (QTLs)approach can be used in a marker-assisted breeding program to identifyalleles of or mutations in the HIO32.3 gene or orthologs of HIO32.3 thatmay confer altered oil content (see Bert et al., Theor Appl Genet. 2003June; 107(1):181-9; and Lionneton et al, Genome. 2002 December;45(6):1203-15). Thus, in a further aspect of the invention, a HIO32.3nucleic acid is used to identify whether a plant having altered oilcontent has a mutation in endogenous HIO32.3 or has a particular allelethat causes altered oil content.

While the invention has been described with reference to specificmethods and embodiments, it will be appreciated that variousmodifications and changes may be made without departing from theinvention. All publications cited herein are expressly incorporatedherein by reference for the purpose of describing and disclosingcompositions and methodologies that might be used in connection with theinvention. All cited patents, patent applications, and sequenceinformation in referenced public databases are also incorporated byreference.

EXAMPLES Example 1 Generation of Plants with a HIO32.2 Phenotype byTransformation with an Activation Tagging Construct

Mutants were generated using the activation tagging “ACTTAG” vector,pSKI015 (GI#6537289; Weigel D et al., 2000). Standard methods were usedfor the generation of Arabidopsis transgenic plants, and wereessentially as described in published application PCT WO183697. Briefly,T0 Arabidopsis (Col-0) plants were transformed with Agrobacteriumcarrying the pSKI015 vector, which comprises T-DNA derived from theAgrobacterium Ti plasmid, an herbicide resistance selectable markergene, and the 4X CaMV 35S enhancer element. Transgenic plants wereselected at the T1 generation based on herbicide resistance. T2 seed wascollected from T1 plants and stored in an indexed collection, and aportion of the T2 seed was accessed for the screen.

Quantitative determination of seed fatty acid content was performedusing the following methods. A sample of 15 to 20 T2 seeds from eachline tested, which generally contained homozygous insertion, homozygouswild-type, and heterozygous genotypes in a standard 1:1:2 ratio, wasmassed on UMT-2 ultra-microbalance (Mettler-Toledo Co., Ohio, USA) andthen transferred to a glass extraction vial. Lipids were extracted fromthe seeds and trans-esterified in 500 ul 2.5% H₂SO₄ in MeOH for 3 hoursat 80° C., following the method of Browse et al. (Biochem J 235:25-31,1986) with modifications. A known amount of heptadecanoic acid wasincluded in the reaction as an internal standard. 750 ul of water and400 ul of hexane were added to each vial, which was then shakenvigorously and allowed to phase separate. Reaction vials were loadeddirectly onto GC for analysis and the upper hexane phase was sampled bythe autosampler. Gas chromatography with Flame Ionization detection wasused to separate and quantify the fatty acid methyl esters. Agilent 6890Plus GC's were used for separation with Agilent Innowax columns (30m×0.25 mm ID, 250 um film thickness). The carrier gas was Hydrogen at aconstant flow of 2.5 ml/minute. 1 ul of sample was injected in splitlessmode (inlet temperature 220° C., Purge flow 15 ml/min at 1 minute). Theoven was programmed for an initial temperature of 105° C., initial time0.5 minutes, followed by a ramp of 60° C. per minute to 175° C., a 40°C./minute ramp to 260° C. with a final hold time of 2 minutes. Detectionwas by Flame Ionization (Temperature 275° C., Fuel flow 30.0 ml/min,Oxidizer 400.0 ml/min). Instrument control and data collection andanalysis were monitored using the Millennium Chromatography ManagementSystem (Version 3.2, Waters Corporation, Milford, Mass.). Integrationand quantification were performed automatically, but all analyses weresubsequently examined manually to verify correct peak identification andacceptable signal to noise ratio before inclusion of the derived resultsin the study.

The ACTTAG line designated WO00082263 was identified as having a highoil phenotype. Specifically, oil constituted 36.3% of seed mass (w/w)compared to an average oil content of 27.0% of other ACTTAG lines grownand analyzed in the same conditions (e.g., reference lines). Reanalysisof the same seed was performed in duplicate. This analysis confirmed anincrease in oil content relative to controls. It was concluded that thepresence of the ACTTAG locus can increase seed oil content between 7%and 11% relative to controls. It is determined that the high oilphenotype is dominant based on oil content of seeds from genotypedindividuals.

Example 2 Characterization of the T-DNA Insertion in Plants Exhibitingthe Altered Oil Content Phenotype

We performed standard molecular analyses, essentially as described inpatent application PCT WO0183697, to determine the site of the T-DNAinsertion associated with the altered oil content phenotype. Briefly,genomic DNA was extracted from plants exhibiting the altered oil contentphenotype. PCR, using primers specific to the pSKI015 vector, confirmedthe presence of the 35S enhancer in plants from line W000082263, andSouthern blot analysis verified the genomic integration of the ACTTAGT-DNA and showed the presence of a single T-DNA insertion in thetransgenic line.

Plasmid rescue was used to recover genomic DNA flanking the T-DNAinsertion, which was then subjected to sequence analysis using a basicBLASTN search and/or a search of the Arabidopsis Information Resource(TAIR) database (publicly available at the Arabidopsis InformationResource website). There was sequence identity to BAC clone T23J7(GI#4741184), chromosome 3. Sequences from nucleotides 16030-16423 and16443-17200 were recovered, placing the left border junction upstream ofnucleotide 16423 (GI#4741184) and downstream of nucleotide 15443. Thesequences from nucleotides 15424-16442 of BAC clone T23J7, chromosome 3(GI#4741184) are deleted in the mutant chromosome.

To determine whether the T-DNA insertion causes the high seed oilphenotype co-segregation of the high seed oil phenotype and the presenceof the T-DNA was tested. Eighteen T2 plants were grown to maturity andseed harvested from these plants was used to determine the seed oilphenotype. The seed oil content from these was determined as describedin Example 1. The genotype of the T2 seed was inferred by analyzing theT3 seed for the presence or absence of the T-DNA at the site of theinsertion by PCR using primers that are specific to the correspondinggenomic region and the T-DNA. The results show that the average oilcontent of T3 seed containing the T-DNA insert was higher than thosefamilies lacking the insert. T2 individuals homozygous for the T-DNA atthis locus produced seed with an oil content of 107.5% compared toprogeny lacking the T-DNA. T2 individuals hemizygous for these lociproduced seed with an oil content of 112.7% compared to progeny lackingthe T-DNA. Because the homozygotes and hemizygotes for the high oil locidisplay a similar increase in oil content, we conclude that the T-DNA islinked with the high oil phenotype and the phenotype is caused by adominant mutation.

Sequence analysis revealed that the left border of the T-DNA insert wasabout 954 base pairs 3′ of the translation start site of the gene whosenucleotide sequence is presented as SEQ ID NO: 1 which we designatedHIO32.2.

Example 3 Analysis of Arabidopsis HIO32.2 Sequence

Sequence analyses were performed with BLAST (Altschul et al., 1997, J.Mol. Biol. 215:403-410), PFAM (Bateman et al., 1999, Nucleic Acids Res27:260-262), PSORT (Nakai K, and Horton P, 1999, Trends Biochem Sci24:34-6), and/or CLUSTAL (Thompson J D et al, 1994, Nucleic Acids Res22:4673-4680).

The TAIR prediction of At3g47710 corresponds to amino acid residues15-92 of Genbank prediction (gi|22331644). In neither cases is theresidue immediately follows the translation initiation codon (ATG) aguanosine (G), which is typical for most genes in Arabidopsis. It islikely that the predicted gene structure for At3g47710 has an atypicalresidue (thymidine, T) following the translation initiation codon (ATG).This proposal is supported by the fact that three Arabidopsis homologuesof At3g47710 (At1g74500, At5g39860 and gi|21617952) whose genestructures are supported by cDNAs have the sequence ATGT at theinitiation codons.

BLASTN Against ESTs:

No Arabidopsis EST shows exact match to the candidate gene At3g47710.However, ESTs corresponding to Arabidopsis homologues of At3g47710 canbe identified. These genes are described in the BLASTP analysis below.

There are also other similar plant ESTs showing similarity to At3g47700.If possible, ESTs contigs of each species were made. The best hit foreach of the following species are listed below and included in the“Orthologue Table” below: Glycine max, Lycopersicon esculentun,Gossypium arboreum, Zea mays, Oryza sativa, Triticum aestivum, Solanumtuberosum.

1. One EST contig from soybean (Glycine max)

>gi|16344264|gb|BI969859.1|BI969859 GM830009A23D05

>gi|16344265|gb|BI969860.1|BI969860 GM830009A23D06

>gi|10848131|gb|BF070679.1|BF070679 st23h10.y1

>gi|19345905|gb|BM890785.1|BM890785 sam07g12.y1

>gi|7925404|gb|AW831369.1|AW831369 sm24d03.y1

Score=310 (114.2 bits), Expect=2.8e-33

These ESTs were isolated from mixed tissues of various developmentalstages or from germinating shoots.

The contigged sequence is presented as SEQ ID NO:3 below.

2. One EST from wheat (Triticum aestivum)

>gi|201034721|dbj|BJ282051.1|BJ282051 BJ282051 from an unpublished cDNAlibrary.

Score=286 (105.7 bits), Expect=9.9e-31

The EST described above (gi|20103472) encompasses the following EST

>gi|20106345|dbj|BJ287183.1|BJ287183 BJ287183 from an unpublished cDNAlibrary.

3. One EST from tomato (Lycopersicon esculentum)

>gi|7409117|gb|AW647879.1|AW647879 EST326333 tomato germinatingseedlings, TAMU library

Score=278 (102.9 bits), Expect=6.9e-30

4. One EST from potato (Solanum tuberosum)

>gi|21375581|gb|BQ516712.1|BQ516712 EST624127 from a library constructedwith mixed tomato tissues

Score=265 (98.3 bits), Expect=1.7e-28

The EST described above (gi|20103472) encompasses the following EST

>gi|21375582|gb|BQ516713.1|BQ516713 EST624128 from a library constructedwith mixed tomato tissues

5. One EST from rice (Oryza sativa)

>gi|8334893|gb|BE039877.1|BE039877 OC09D12 OC Oryza sativa cDNA 5′, mRNAfrom 1 week old roots

Score=228 (85.3 bits), Expect=1.4e-24

6. One EST from maize (Zea mays)

>gi|18173123|gb|BM348511.1|BM348511 MEST292-A06.T3 ISUM5-RNA Zea mayscDNA clone isolated from a B73 Maize library constructed with tissuesfrom various stages and treated with a variety of hormones

Score=202 (76.2 bits), Expect=7.9e-22,

7. One EST from tree cotton (Gossypium arboreum)

>gi|18098731|gb|BM357985.1|BM357985 GA_Ea0003E23r Gossypium arboreum7-10 dpa fiber library

Score=197 (74.4 bits), Expect=2.7e-21

BLASTP against all.aa results:

At3g47710 has homology to a small number of plant proteins. The top 7BLAST hits are listed below and are included in the “Orthologue Table”below.

1. Itself (3 redundant entries)

>gi|22331645|ref|NP_(—)190355.2| bHLH protein; protein id: At3g47710.1[Arabidopsis thaliana]

Score=123 bits (309), Expect=5e-28

The following 2 sequences are redundant entries of At3g47710 based onGenbank annotation. The predicted gene structure for At3g47710 fromgi|7487374 and gi|4741188 corresponds to amino acid residues 15-92 ofgi|22331645.

>gi|7487374|pir∥T07710 hypothetical protein T23J7.40—Arabidopsisthaliana

>gi|4741188|emb|CAB41854.1| hypothetical protein [Arabidopsis thaliana]

Score=111 bits (277), Expect=2e-24

2. At1g74500 from Arabidopsis (4 redundant entries)

>gi|15221264|ref|NP_(—)177590.1| bHLH protein; protein id: At1g74500.1,supported by cDNA: 519. [Arabidopsis thaliana]

>gi|25406349|pir∥A96774 probable DNA-binding protein F1M20.18[imported]—Arabidopsis thaliana

>gi|12324788|gb|AAG52350.1|AC01765_(—)2 putative DNA-binding protein;54988-54618 [Arabidopsis thaliana]

>gi|21593858|gb|AAM65825.1| putative DNA-binding protein [Arabidopsisthaliana]

Score=78.2 bits (191), Expect=2e-14

3. A DNA-binding protein-like protein from chromosome 3 of Arabidopsis(2 redundant entries in Genbank)

>gi|21617952|gb|AAM67002.1| DNA-binding protein-like [Arabidopsisthaliana]

Score=73.9 bits (180), Expect=4e-13

This is a gene represented by a cDNA that is mapped to chromosome 3 ofArabidopsis. There is no TAIR annotation of this gene.

The following entry is likely to be a redundant entry of gi|21617952,because BLASTN shows that the sequences gi|21617952 and gi|9294226 arelocalized to the same place in the Arabidopsis genome. The sequencesgi|21617952 and gi|9294226 differ by a few amino acids. These are likelyto be sequencing errors or single nucleotide polymorphisms with littleor no effect on activity.

>gi|9294226|dbj|BAB02128.1| DNA-binding protein-like [Arabidopsisthaliana]

Score=74.7 bits (182), Expect=2e-13

4. At5g39860 of Arabidopsis (3 redundant entries in Genbank)

>gi|15242499|ref|NP_(—)198802.1| bHLH protein; protein id: At5g39860.1[Arabidopsis thaliana]

>gi|10176978|dbj|BAB10210.1| DNA-binding protein-like [Arabidopsisthaliana]

>gi|21593819|gb|AAM65786.1| DNA-binding protein-like [Arabidopsisthaliana]

Score=70.9 bits (172), Expect=4e-12

5. At5g15160 of Arabidopsis (3 redundant entries in Genbank)

>gi|15242227|ref|NP_(—)197020.1| bHLH protein; protein id: At5g1560.1[Arabidopsis thaliana]

>gi|11357892|pir∥T49951 hypothetical protein F8M21.50—Arabidopsisthaliana

>gi|7671485|emb|CAB89326.1| putative protein [Arabidopsis thaliana]

Score=69.7 bits (169), Expect=8e-12

6. A hypothetical protein from rice (Oryza sativa)

>gi|21671920|gb|AAM74282.1|AC083943_(—)22 Hypothetical protein similarto putative DNA binding proteins [Oryza sativa (japonicacultivar-group)]

Score=54.3 bits (129), Expect=4e-07

7. A putative protein from rice (Oryza sativa)

>gi|10241623|emb|CAC09463.1| putative DNA binding protein [Oryza sativa(indica cultivar-group)]

Score=53.5 bits (127), Expect=6e-07

% ID to Score(s) Coordinates NEWGENE motif Ortholog % ID to (BLAST, ofprotein or to Pfam/other Gene Name Species GI # HIO32.2 Clustal, etc.)motif (s) consensus A Oryza >gi|21671920 Length = 88 BLASTP SM00353:SM00353: hypothetical saliva (japonica Identities = 44/93 (47%), Score =aa 4-56 Score = −0.5, E protein from cultivar-group) Positives = 65/93(69%) 54.3 bits (129), PF00010: value = 0.13 rice Expect = 4e−07 aa 5-61PF00010: Score = 11.4, E value = 0.0018 A putative Oryza >gi|10241623Length = 104 BLASTP SM00353: SM00353: protein from sativa (indicaIdentities = 53/102 (51%), Score = aa 26-77 Score = 13.7, E ricecultivar-group) Positives = 70/102 (68%) 53.5 bits (127), PF00010: value= 0.0097 Expect = 6e−07 aa 23-72 PF00010: Score = 7.9, E value = 0.017An EST Glycine >gi|16344264 Partial BLASTN SM00353: SM00353: contig frommax >gi|16344265 sequence, using Score = aa 18-73 Score = 16.2, Esoybean >gi|10848131 reading frame + 1 310 (114.2 bits), PF00010: value= 0.0049 >gi|19345905 Length = 99 Expect = 2.8e−33, aa 18-68PF00010: >gi|7925404 Identities = 66/93 (70%), P = 2.8e−33 Score = 11.0,E Positives = 83/93 (89%) value = 0.0076 A wheat Triticum >gi|20103472Partial BLASTN SM00353: SM00353: EST aestivum sequence, using Score = aa41-90 Score = 11.1, E reading frame + 3 286 (105.7 bits), PF00010: value= 0.019 Length = 116 Expect = 9.9e−31, aa 29-85 PF00010: Identities =64/93 (68%), P = 9.9e−31 Score = 0.4, E Positives = 79/93 (84%) value =0.11 A tomato Triticum >gi|7409117 Partial BLASTN SM00353: SM00353: ESTaestivum sequence, using Score = aa 10-64 Score = 12.6, E readingframe + 2 278 (102.9 bits), PF00010: value = 0.013 Length = 91 Expect =6.9e−30, aa 9-59 PF00010: Identities = 65/93 (69%), P = 6.9e−30 Score =6.9, E Positives = 77/93 (82%) value = 0.021 A potatoSolanum >gi|21375581 Partial BLASTN SM00353: SM00353: EST tuberosumsequence, using Score = aa 16-71 Score = 9.1, E reading frame + 1 265(98.3 bits), PF00010: value = 0.033 Length = 109 Expect = 1.7e−28, aa13-66 PF00010: Identities = 61/95 (64%), P = 1.7e−28 Score = 1.9, EPositives = 77/95 (81%) value = 0.074 A rice Oryza >gi|8334893 PartialBLASTN SM00353: SM00353: EST from root sativa sequence, using Score = aa2-49 Score = 7.9, E reading frame + 2 228 (85.3 bits), PF00010: value =0.043 Length = 76 Expect = 1.4e−24, aa 1-44 PF00010: Identities = 52/77(67%), P = 1.4e−24 Score = −1.0, E Positives = 64/77 (83%) value = 0.14A maize Zea mays >gi|18173123 Partial BLASTN SM00353: SM00353: ESTsequence, using Score = aa 42-94 Score = 12.7, E reading frame − 2 202(76.2 bits), PF00010: value = 0.013 Length = 121 Expect = 7.9e−22, aa37-89 PF00010: Identities = 54/104 (51%), P = 7.9e−22 Score = 5.2, EPositives = 74/104 (71%) value = 0.032 A cotton Gossypium >gi|18098731Partial BLASTN SM00353: SM00353: fiber EST arboreum sequence, usingScore = aa 7-58 Score = 26.3, E reading frame − 2 197 (74.4 bits),PF00010: value = 7.6e−05 Length = 85 Expect = 2.7e−21, aa 4-53 PF00010:Identities = 41/83 (49%), P = 2.7e−21 Score = 14.4, E Positives = 62/83(74%) value = 0.0033 Closest Arabidopsis homologs: At1g74500Arabidopsis >gi|15221264 Length = 93 BLASTP SM00353: SM00353:thaliana >gi|25406349 Identities = 58/93 (62%), Score = aa 16-67 Score =21.7, E >gi|12324788 Positives = 77/93 (82%) 78.2 bits (191), PF00010:value = 0.0011 >gi|21593858 Expect = 2e−14 aa 7-62 PF00010: Score =−14.3, E value = 0.033 A DNA- Arabidopsis >gi|21617952 Length = 92BLASTP SM00353: SM00353: binding protein- thaliana Identities = 57/93(61%), Score = aa 10-65 Score = 7.8, E like protein Positives = 74/93(79%) 73.9 bits (180), PF00010: value = 0.048 Expect = 4e−13 aa 6-60PF00010: Score = −0.9, E value = 0.15 At5g39860 Arabidopsis >gi|15242499Length = 92 BLASTP SM00353: SM00353: thaliana >gi|10176978 Identities =55/93 (59%), Score = aa 10-65 Score = 3.6, E >gi|21593819 Positives =71/93 (76%) 70.9 bits (172), PF00010: value = 0.15 Expect = 4e−12 aa5-60 PF00010: Score = −0.3, E value = 0.13 At5g15160Arabidopsis >gi|15242227 Length = 94 BLASTP SM00353: SM00353:thaliana >gi|11357892 Identities = 53/91 (58%), Score = aa 6-61 Score =4.8, E >gi|7671485 Positives = 71/91 (78%) 69.7 bits (169), PF00010:value = 0.036 Expect = 8e−12 aa 11-66 PF00010: Score = −9.6, E value =0.029

At3g47710 is a non-secretory protein that lacks transmembrane domain(predicted by TMHMM) and signal peptide (predicted by SignalP). PSORT2predicts At3g47710 to be localized to the mitochondria (64%mitochondrial, 24% nuclear, 8% cytoplasmic, 4% peroxisomal). However,Predator analysis gives contradictory results and suggests thatAt3g47710 is localized to neither the mitochondria nor the plastid (5%mitochondrial, 0% plastid). Therefore,

Pfam analysis showed that At3g47710 shows weak homology to thehelix-loop-helix DNA-binding domain (PF00010, SM00353). The basichelix-loop-helix proteins (bHLH) are a group of eukaryotic transcriptionfactors that have diverse functions. These transcription factors arecharacterized by a highly conserved bHLH domain that mediates DNAbinding and dimerisation with other proteins (Littlewood and Evan, 1995Protein Profile 2,621-702).

A number of Arabidopsis bHLH proteins have been characterized and shownto be important for diverse functions such as development of carpelmargin, fruit dehiscence, light signal transduction, root hairdevelopment and trichome development (Heisler et al., 2001 Development.128, 1089-1098; Rajani and Sundaresan, 2001, Curr Biol. 11, 1914-1922;Huq and Quail, 2002 EMBO J. 21, 2441-2450; Wada et al., 2002,Development. 129, 5409-5419; Smolen et al., 2002, Genetics. 161,1235-1246; Sawa, 2002, DNA Res. 9, 31-34). It should be noted thatBLASTP analysis of At3g47710 did not identify functionally characterizedbHLH proteins in Arabidopsis, suggesting that At3g47710 is a distantfamily member to these bHLH proteins. Nonetheless, it is conceivablethat over-expression of the transcription factor At3g47710 may increaseseed oil content by altering expression of downstream genes in eitherthe nucleus or the mitochondria.

seq- seq- hmm- hmm- E- Model Domain f t f t score value SM00353 1/1 1666 . . . 1 61 [ ] 9.8 0.028 PF00010 1/1 16 61 . . . 1 53 [ ] 1.9 0.073

Example 4 Confirmation of Phenotype/Genotype Association

RT-PCR analysis showed that the HIO32.2 gene was over-expressed inplants from the line displaying the HIO32.2 phenotype. Specifically, RNAwas extracted from rosette leaves and/or siliques of plants exhibitingthe HIO32.2 phenotype collected at a variety of developmental stages andpooled. RT-PCR was performed using primers specific to the sequencepresented as SEQ ID NO:1, to other predicted genes in the vicinity ofthe T-DNA insertion, and to a constitutively expressed actin gene(positive control). The results showed that in plants displaying theHIO32.2 phenotype, mRNA for the HIO32.2 gene is up-regulated in leavesand down-regulated in siliques.

The dominant inheritance pattern of the HIO32.2 phenotype is confirmedthrough genetic analysis. In general, genetic analysis involves theproduction and analysis of F1 hybrids. Typically, F1 crosses are carriedout by collecting pollen from T2 plants, which is used to pollinate wildtype plants. Such crosses are carried out by taking about 4 flowers fromeach selected individual plants, and using the T2 flower as the malepollen donor and flowers of the wild type plants as the female. 4-5crosses are done for an individual of interest. Seed formed from crossesof the same individual are pooled, planted and grown to maturity as F1hybrids.

Example 5 Recapitulation of the High Oil Phenotype

To confirm whether over-expression of At3g47710 causes a high seed oilphenotype, oil content in seeds from transgenic plants over-expressingthis gene was compared with oil content in seeds from non-transgeniccontrol plants. To do this, At3g47710 was cloned into a planttransformation vector behind the strong constitutive CsVMV promoter andtransformed into Arabidopsis plants using the floral dip method. Theplant transformation vector contains the nptII gene, which providesresistance to kanamycin, and serves as a selectable marker. Seed fromthe transformed plants were plated on agar medium containing kanamycin.After 7 days, transgenic plants were identified as healthy green plantsand transplanted to soil. Non-transgenic control plants were germinatedon agar medium, allowed to grow for 7 days and then transplanted tosoil. Twenty-two transgenic seedlings and 10 non-transgenic controlplants were transplanted to random positions in the same 32 cell flat.The plants were grown to maturity, allowed to self-fertilize and setseed. Seed was harvested from each plant and its oil content estimatedby Near Infrared (NIR) Spectroscopy using methods described below.

NIR infrared spectra were captured using a Bruker 22 N/F near infraredspectrometer. Bruker Software was used to estimate total seed oil andtotal seed protein content using NIR data from the samples and referencemethods according to the manufacturer's instructions. An oil contentpredicting calibration was developed following the general method ofAOCS Procedure Am1-92, Official Methods and Recommended Practices of theAmerican Oil Chemists Society, 5th Ed., AOCS, Champaign Ill.). Thecalibration allowing NIR predictions of Crude Oil Crude Oil ASE (RenOil, Accelerated Solvent Extraction) was developed.

The effect of over-expression of At3g47710 on seed oil has been testedin five experiments. In four experiments, the plants over-expressingAt3g47710 had higher seed oil content than the control plants grown inthe same flat. Across the experiments, the average seed oil content ofplants over-expressing At3g47710 was 3.5% greater than the untransformedcontrols. The in seed oil content in plants over-expressing At3g47710was significantly greater than non-transgenic control plants (two-wayANOVA; P=0.0066), see Table 1.

TABLE 1 Relative Experi- Predicted value ment Plant ID Transgene averageaverage 1 DX02948001 CsVMV::HIO32.2 31.2429 93.4123 1 DX02948003CsVMV::HIO32.2 34.8987 104.3425 1 DX02948004 CsVMV::HIO32.2 32.991598.6401 1 DX02948005 CsVMV::HIO32.2 35.0001 104.6456 1 DX02948006CsVMV::HIO32.2 36.5424 109.2568 1 DX02948007 CsVMV::HIO32.2 36.7931110.0065 1 DX02948008 CsVMV::HIO32.2 36.4274 108.9132 1 DX02948009CsVMV::HIO32.2 35.0468 104.7852 1 DX02948011 CsVMV::HIO32.2 34.2905102.5239 1 DX02948013 CsVMV::HIO32.2 33.4158 99.9088 1 DX02948014CsVMV::HIO32.2 34.1728 102.1721 1 DX02948015 CsVMV::HIO32.2 31.657994.6528 1 DX02947001 None 35.6386 106.5548 1 DX02947002 None 34.0045101.6688 1 DX02947003 None 32.578 97.4039 1 DX02947004 None 33.5877100.4227 1 DX02947005 None 33.0303 98.7563 1 DX02947006 None 34.3919102.8273 1 DX02947007 None 33.1974 99.2558 1 DX02947008 None 31.14293.1104 2 Z003900001 CsVMV::HIO32.2 33.6896 106.2791 2 Z003900002CsVMV::HIO32.2 33.3141 105.0947 2 Z003900003 CsVMV::HIO32.2 35.6526112.4718 2 Z003900004 CsVMV::HIO32.2 27.8247 87.7776 2 Z003900005CsVMV::HIO32.2 33.1646 104.6231 2 Z003900006 CsVMV::HIO32.2 37.0337116.8286 2 Z003900008 CsVMV::HIO32.2 34.6903 109.4361 2 Z003900009CsVMV::HIO32.2 30.981 97.7346 2 Z003900010 CsVMV::HIO32.2 39.056123.2082 2 Z003900011 CsVMV::HIO32.2 37.1317 117.1378 2 Z003900013CsVMV::HIO32.2 32.409 102.2392 2 Z003900014 CsVMV::HIO32.2 36.3444114.6543 2 Z003900015 CsVMV::HIO32.2 36.8128 116.1318 2 Z003900017CsVMV::HIO32.2 38.4427 121.2736 2 Z003900018 CsVMV::HIO32.2 33.6492106.1516 2 Z003900020 CsVMV::HIO32.2 30.8397 97.2888 2 Z003900021CsVMV::HIO32.2 35.9108 113.2864 2 Z003890001 None 36.4237 114.9044 2Z003890003 None 32.4789 102.4599 2 Z003890004 None 33.9902 107.2273 2Z003890005 None 29.5647 93.2666 2 Z003890006 None 31.4827 99.3173 2Z003890007 None 27.8273 87.7857 2 Z003890008 None 26.2013 82.6561 2Z003890009 None 34.3203 108.269 2 Z003890010 None 33.0031 104.1136 3Z003978002 CsVMV::HIO32.2 33.4407 97.8939 3 Z003978003 CsVMV::HIO32.236.4683 106.7568 3 Z003978004 CsVMV::HIO32.2 34.9617 102.3464 3Z003978005 CsVMV::HIO32.2 34.7488 101.7233 3 Z003978007 CsVMV::HIO32.231.0467 90.8856 3 Z003978008 CsVMV::HIO32.2 36.6832 107.3859 3Z003978010 CsVMV::HIO32.2 34.8046 101.8866 3 Z003978011 CsVMV::HIO32.226.9809 78.9835 3 Z003978012 CsVMV::HIO32.2 32.4106 94.8785 3 Z003978014CsVMV::HIO32.2 35.5825 104.1638 3 Z003978015 CsVMV::HIO32.2 33.929599.3247 3 Z003978016 CsVMV::HIO32.2 34.9754 102.3866 3 Z003978017CsVMV::HIO32.2 33.5072 98.0887 3 Z003978018 CsVMV::HIO32.2 35.3474103.4756 3 Z003978019 CsVMV::HIO32.2 33.4171 97.8248 3 Z003978022CsVMV::HIO32.2 31.2548 91.4948 3 Z003994001 None 35.8594 104.9744 3Z003994002 None 34.3685 100.61 3 Z003994003 None 34.7967 101.8634 3Z003994004 None 36.266 106.1646 3 Z003994005 None 30.8633 90.3488 3Z003994006 None 32.8165 96.0666 3 Z003994007 None 36.5016 106.8543 3Z003994008 None 34.9869 102.4202 3 Z003994009 None 30.7767 90.0952 3Z003994010 None 34.3659 100.6024 4 DX06941001 CsVMV::HIO32.2 33.9232104.5537 4 DX06941002 CsVMV::HIO32.2 33.6401 103.6812 4 DX06941003CsVMV::HIO32.2 35.7409 110.1562 4 DX06941004 CsVMV::HIO32.2 31.943998.4536 4 DX06941005 CsVMV::HIO32.2 31.3507 96.6252 4 DX06941006CsVMV::HIO32.2 32.5406 100.2926 4 DX06941007 CsVMV::HIO32.2 33.1874102.286 4 DX06941008 CsVMV::HIO32.2 34.0966 105.0881 4 DX06941009CsVMV::HIO32.2 34.7597 107.132 4 DX06941010 CsVMV::HIO32.2 32.8897101.3686 4 DX06941011 CsVMV::HIO32.2 34.0769 105.0275 4 DX06941012CsVMV::HIO32.2 32.9075 101.4232 4 DX06941013 CsVMV::HIO32.2 36.1765111.4985 4 DX06941014 CsVMV::HIO32.2 34.3151 105.7617 4 DX06941015CsVMV::HIO32.2 31.8685 98.2211 4 DX06941016 CsVMV::HIO32.2 32.227999.3287 4 DX06941017 CsVMV::HIO32.2 35.2172 108.542 4 DX06941018CsVMV::HIO32.2 30.4763 93.9302 4 DX06941019 CsVMV::HIO32.2 30.57394.2281 4 DX06941020 CsVMV::HIO32.2 32.4645 100.0581 4 DX06941021CsVMV::HIO32.2 36.0598 111.139 4 DX06941022 CsVMV::HIO32.2 31.483497.0342 4 DX06959001 None 32.2856 99.5067 4 DX06959002 None 31.187596.122 4 DX06959004 None 34.4381 106.1409 4 DX06959005 None 32.177399.1728 4 DX06959006 None 33.4702 103.1577 4 DX06959007 None 33.1607102.2038 4 DX06959008 None 33.1406 102.1416 4 DX06959009 None 29.774391.7666 4 DX06959010 None 32.3769 99.7879 5 DX06942001 CsVMV::HIO32.232.4339 104.7283 5 DX06942002 CsVMV::HIO32.2 32.5468 105.0928 5DX06942003 CsVMV::HIO32.2 31.6804 102.2953 5 DX06942004 CsVMV::HIO32.231.2728 100.9792 5 DX06942005 CsVMV::HIO32.2 34.0027 109.7938 5DX06942006 CsVMV::HIO32.2 31.9079 103.0297 5 DX06942007 CsVMV::HIO32.231.0441 100.2405 5 DX06942008 CsVMV::HIO32.2 30.5782 98.7361 5DX06942009 CsVMV::HIO32.2 35.4122 114.3452 5 DX06942010 CsVMV::HIO32.232.6468 105.4156 5 DX06942011 CsVMV::HIO32.2 35.5334 114.7364 5DX06942012 CsVMV::HIO32.2 31.0164 100.1512 5 DX06942014 CsVMV::HIO32.229.8991 96.5435 5 DX06942015 CsVMV::HIO32.2 33.7687 109.0383 5DX06942016 CsVMV::HIO32.2 31.959 103.1948 5 DX06942017 CsVMV::HIO32.232.7295 105.6828 5 DX06942018 CsVMV::HIO32.2 32.3263 104.3809 5DX06942019 CsVMV::HIO32.2 33.0989 106.8755 5 DX06942020 CsVMV::HIO32.229.9662 96.7602 5 DX06960001 None 28.8877 93.2776 5 DX06960002 None33.7045 108.8311 5 DX06960003 None 29.816 96.275 5 DX06960004 None32.7046 105.6022 5 DX06960005 None 31.3342 101.1775 5 DX06960006 None31.0325 100.2031 5 DX06960007 None 32.8594 106.1021 5 DX06960008 None28.4125 91.7432 5 DX06960009 None 30.4112 98.1969 5 DX06960010 None30.5333 98.5913

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1. A transgenic plant comprising a heterologous polynucleotide thatencodes a HIO32.2 polypeptide having an amino acid sequence at least 95%identical to the amino acid sequence of SEQ ID NO: 2 and conveying ahigh oil phenotype on a plant when expressed in said plant, whereby thetransgenic plant has at least 3.5% greater seed oil content relative toa plant of the same species that does not comprise the heterologouspolynucleotide.
 2. The transgenic plant of claim 1, wherein theheterologous polynucleotide encodes a HIO32.2 polypeptide having theamino acid sequence of SEQ ID NO:
 2. 3. The transgenic plant of claim 1,which is selected from the group consisting of rapeseed, soy, corn,sunflower, cotton, cocoa, safflower, oil palm, coconut palm, flax,castor and peanut.
 4. The transgenic plant of claim 1, furthercomprising a heterologous constitutive promoter operatively linked tothe heterologous polynucleotide.
 5. A plant part obtained from the plantaccording to claim
 1. 6. The plant part of claim 5, wherein the plantpart has at least 3.5% greater oil content relative to an equivalentplant part obtained from a plant of the same species that does notcomprise the heterologous polynucleotide.
 7. A meal, feed, or foodcomprising a plant part obtained from the plant of claim 1, wherein themeal, feed, or food has at least 3.5% greater oil content relative tomeal feed or food comprising a plant part from a plant of the samespecies that does not comprise the heterologous polynucleotide.
 8. Theplant part of claim 5, which is a seed.
 9. The seed of claim 8, whereinthe seed has at least 3.5% greater oil content relative to seed from aplant of the same species that does not comprise the heterologouspolynucleotide.
 10. A meal, feed, or food comprising the seed of claim8, wherein the meal, feed, or food has at least 3.5% greater oil contentrelative to meal, feed, or food comprising seed from a plant of the samespecies that does not comprise the heterologous polynucleotide.
 11. Amethod of producing oil comprising growing the transgenic plant of claim1 and recovering oil from said plant.
 12. The method of claim 11,wherein the oil is recovered from a seed of the plant.
 13. A feed, meal,grain, food, or seed comprising a polypeptide: encoded by the nucleicacid sequence as set forth in SEQ ID NO: 1; having the amino acidsequence set forth in SEQ ID NO: 2; or having an amino acid sequence atleast 95% identical to the amino acid sequence of SEQ ID NO: 2, whereinthe feed, meal, grain, food, or seed has at least 3.5% greater oilcontent relative to meal, grain, food, or seed that does not comprisethe polypeptide.
 14. A transgenic plant with a high oil phenotype madeby a method comprising: introducing into progenitor cells of the plant aheterologous constitutive promoter operatively linked to a heterologouspolynucleotide that encodes a HIO32.2 polypeptide, to producetransformed cells overexpressing the HIO32.2 polypeptide, wherein theHIO32.2 polypeptide has an amino acid sequence at least 95% identical tothe amino acid sequence of SEQ ID NO: 2 and conveys a high oil phenotypeon a plant when expressed in said plant; growing the transformedprogenitor cells to produce a transgenic plant; and identifying the highoil phenotype in said transgenic plant by measuring oil content of thetransgenic plant and selecting a transgenic plant that possesses atleast 3.5% greater seed oil content relative to a plant of the samespecies that does not comprise the heterologous constitutive promoteroperatively linked to the heterologous polynucleotide; thereby producingthe transgenic plant with a high oil phenotype.
 15. The transgenic plantof claim 14 wherein the HIO32.2 polypeptide has the amino acid sequenceof SEQ ID NO: 2.